Our overall goal is to understand how biological systems that contain paramagnetic centers work. We probe these paramagnetic centers to determine local liganding structure, unpaired electron distribution, evidence for protein-induced perturbation, and kinetic change in the formation and interconversion of paramagnetic species. Our investigation is through the techniques of electron nuclear double resonance (ENDOR) and electron paramagnetic resonance (EPR), including rapid-mix flow and stopped-flow EPR which have been recently developed. Besides EPR-ENDOR, we bring complementary spectroscopic, kinetic, and biochemical methods as needed. The systems to be studied are: 1. Metal centers of bioenergetic importance that couple oxygen consumption and electron transport in terminal cytochrome c and quinol oxidases. The purpose will be to understand intermediates of oxygen chemistry at the heme-copper center and perturbing influences on the binuclear CUA center. 2. Stable radical sites of bioenergetic importance that couple electron transfer to the production of ion gradients. The purpose will be to probe protein-radical hydrogen bonding and to understand how the protein perturbs radical spin density and electronic structure. 3. Proteins of denitrification which enzymatically produce nitric oxide from nitrite or sense and bind nitric oxide to protect against nitric oxide toxicity. The active site copper of nitrite reductase will be probed for intermediates of nitrite reduction using cryoreduction techniques. We will next investigate the unusual heme locale of NO-binding cytochrome c. 4. Iron-containing Bleomycin that selectively attacks DNA. We will use ENDOR to explore the interaction and geometry of the bleomycin-target DNA complex, and then, with our newly developed flow and stopped-flow EPR apparatus we will probe the kinetics of peroxyl radical formation catalyzed by bleomycin.